Membrane for use in guided tissue regeneration

ABSTRACT

The invention provides a multi-layer membrane comprising a matrix layer predominantly of collagen II and having an open sponge-like texture, and at least one barrier layer having a close, relatively impermeable texture. Such a membrane is particularly suitable for use in guided tissue regeneration, in particular for use in vivo in the reconstruction of bone or cartilage tissue.

[0001] The present invention concerns a collagen membrane implant foruse in guided tissue regeneration, in particular for use in vivo in thereconstruction of bone or cartilage tissue.

[0002] In tissue regeneration, it has long proved difficult toreconstruct cartilage tissue, such as in cartilage lesions. Cartilageinjuries can occur in any joint though the larger joints, such as theknee and ankle, are most at risk. Such injuries can result from trauma,from degenerative conditions or osteochondritis dissecans. Cartilageinjuries are a principal pathomechanical factor in the development ofarthrosis. The liberation of enzymes leads to an inflammatory process ofthe synovia which in turn leads to abrasion of the cartilage anddestruction of the joint surface. Recent attempts to regeneratearticular cartilage in chondral defects in vivo include implantation ofcultured autogenic articular chondrocytes (CACs). However, thistechnique has had limited success.

[0003] It is now generally accepted that reconstruction of tissuerequires the provision of a matrix to serve as a guide for cells, whichgrow along and between the fibres of the matrix. More recently, the useof CACs seeded in both synthetic and natural resorbable matrices hasbeen proposed. However, attempts to reconstruct cartilage tissue usingmatrices based on polylactic acid, polyglycolic acid and collagen I orIII, have required the matrices to be loaded in vitro with chondrocytesprior to implantation. This gives rise to complications in terms of thesterile culture of the chondrocytes i.e. immunological inflammatoryreactions by giant cells and fibroblastic cells at the interface betweenimplants and tissue.

[0004] WO-A-96/25961 proposes a matrix implant based on collagen IIwhich can be implanted at the in viva site and which relies on thegrowth of native chondrocytes on the surface of the matrix to effectcartilage regeneration. The ability of such a matrix to effect completeregeneration of cartilage tissue is, however, limited.

[0005] There is thus a need for a matrix implant which will permitsuccessful ingrowth of native chondrocytes and thus regeneration ofcartilage tissue following implantation in vivo. We have now found thatcartilage, and ultimately new bone tissue, can be reconstructed by theuse of a cbllagen II matrix which in vivo is shielded not only from thesurrounding connective tissue but also from the underlying bone orcartilage defect. It is envisaged that this may be achieved through theuse of a multi-layer membrane implant which itself is capable ofpreventing the undesired ingrowth of any surrounding tissues into thematrix, or which may be surgically implanted at the site of the defectso as to achieve this effect.

[0006] Viewed from one aspect the invention thus provides a multi-layermembrane comprising a matrix layer predominantly of collagen II andhaving an open sponge-like texture, and at least one barrier layerhaving a close, relatively impermeable texture.

[0007] A particular advantage of the membrane according to the inventionwhen used is that native cells are unable to penetrate or grow into thelayer having a close, relatively impermeable texture.

[0008] Whilst not wishing to be bound by theory, it is now believed thatsuccessful cartilage regeneration requires that the rapid ingrowth notonly of native tissue cells, such as connective tissues, blood vesselsetc., but also of any new bone tissue into the site of the defect beprevented. This may be achieved using a double-layer membrane inaccordance with the invention which serves to shield the collagen matrixfrom the ingrowth of native tissue cells from one side. During surgicalimplantation this may be used in combination with a tissue graft, e.g. aperiosteal graft, effective to prevent the ingrowth of native tissuecells from the opposing side. Thus, for example, a periosteal graft mayinitially be sutured in place such that this provides a covering overthe bone or cartilage defect. A double-layer membrane of the inventionmay then be implanted at the site of the defect such that this lies incontact with the graft and may be arranged in such a way that the matrixlayer faces towards the bone defect. More preferably, a double-layermembrane of the invention is initially implanted at the site of thedefect with the barrier layer facing towards the bone or cartilagedefect. A periosteal graft is then arranged such that this lies incontact with the matrix layer. The graft may be adhered with abiocompatible adhesive such as fibrin glue, or pinned with resorbablepolylactic pins, or if necessary or possible sutured in such a way thatthis then serves to provide an impermeable barrier to the ingrowth ofany surrounding connective tissue.

[0009] In an alternative embodiment of the invention, the membrane.itself may be effective to prevent the ingrowth of any native tissuecells. Thus, viewed from a further aspect the invention provides amembrane comprising at least three layers in which a matrix layer beingpredominantly made from collagen II and having an open sponge-liketexture is provided between two barrier layers having a close,relatively impermeable texture.

[0010] The matrix layer is capable of acting as a medium for theingrowth of native chondrocytes thereby effecting regeneration ofcartilage tissue. However, to further aid in regenerating cartilagetissue the matrix layer may be impregnated with chondrocytes eitherprior to or following implantation in vivo. Whilst the matrix layer maybe impregnated with chondrocytes immediately prior to implantation, e.g.by injection, it is expected that in general the chondrocytes will beintroduced into the matrix layer by direct injection of a suspension ofchondrocytes following implantation. In this way, chondrocytes presentin the matrix layer of the membrane are able to effect regeneration ofcartilage, and ultimately new bone, whilst the membrane at the same timeprevents the ingrowth of other cell types from the surrounding tissues.

[0011] Chondrocytes for use in the invention may be obtained from cellsources which include allogenic or autogenic cells isolated fromarticular cartilage, periosteum and per ichondrium, and mesenchymal(stromal) stem cells from bone marrow. Since allogenic cells carry thepotential for immune response and infectious complications, it ispreferable to isolate the chondrocytes from autogenic cells, especiallyfrom autogenic articular cartilage. Techniques for harvesting cells areknown and include enzymatic digestion or outgrowth culture. Theharvested cells are then expanded in cell culture prior toreintroduction to the body. In general, at least 10⁶, preferably atleast 10⁷ cells should be impregnated into the matrix layer to providefor optimal regeneration of cartilage tissue.

[0012] In general, it is desirable for the matrix layer of the membraneaccording to the invention to contain glycosaminoglycans (GAGs) such ashyaluronic acid, chondroitin 6-sulphate, keratin sulphate, dermatansulphate etc. which serve to provide a natural medium in whichchondrocytes can become embedded and grow. Whilst it is possible toincorporate into the collagen matrix glycosaminoglycans from differentsources which do not necessarily have the same composition, molecularweight and physiological properties as those from cartilage, preferredglycosaminoglycans are those extracted from cartilage itself. Ingeneral, the matrix layer preferably contains from 1 to 10 wt % ofglycosaminoglycans, for example 2 to 6 wt %. Although someglycosaminoglycans may be present in the impermeable layer, the greaterpart will be present in the matrix layer.

[0013] In native collagen tissues GAGs occur, at least in part, as acomponent of proteoglycans (PGs). The use of GAGs in the form of PGs isundesirable in view of potential immunological problems which can becaused by the protein content of the PGs. Preferably, the matrix layeris thus substantially free from any proteoglycans. Conveniently, thismay be achieved by preparing the matrix layer from a mixture of apurified telopeptide-free collagen II material and glycosaminoglycans.

[0014] Other additives which may also be present in the matrix layerinclude, for example, chondronectin, laminin, fibronectin, calciumalginate or anchorin II to assist attachment of the chondrocytes to thecollagen II fibres, and growth factors such as cartilage inducing factor(CIF), insulin-like growth factor (IGF), transforming growth factor β(TGFβ) present as homodimers or heterodimers and bone morphogeneticfactors (BMP) such as native or recombinant human BMP-2, BMP-3(osteogenin), BMP-4 and BMP-7 (OP-1, osteogenetic protein-1). BMP-2affects the two pathways of bone formation independently—the directformation of bone as well as the formation of cartilage which is thenremoved and replaced by bone. Composites of BMPs and collagen includingbone matrix obtained by extraction from cortical bone from varioussources or demineralised bone matrix consist of 90% collagen and 10%non-collagenous proteins (NCP) for BMP activity or for BMP/NCP inducedchondrogenesis. Bone matrix-insoluble collagenous matrix and laminin orfibronectin act as carriers for BMPS. Some growth factors may also bepresent in the impermeable layer. However, the greater part will bepresent in the matrix layer. In general, the membrane contains from 100μg to 5 mg of growth factors.

[0015] As indicated above, the membrane comprises at least two layershaving different structures. Preferably, the barrier layer of themembrane is predominantly made from collagen I and III. Alternatively,this may comprise a synthetic material, e.g. a synthetic resorbablepolymer network optionally coated with a collagen material such as typeI and/or type III collagen.

[0016] Examples of suitable synthetic materials include polyesters,polyglycolic and polylactic acids (PLA) homopolymers and copolymers,glycolide and lactide copolymers, polyorthoesters and polycaprolactones.Many examples of these are openly available, e.g. from BoehringerIngelheim in their RESOMER range. PLA polymers as wax with anappropriate molecular size of ca. 650-1200 and not too rapid adegradation are preferred. A particularly preferred biodegradablepolymer is poly(D,L-lactic acid) in which the ratio of D-lactide toL-lactide is approx. 70:30. An advantage of such synthetic materials isthat these can have high mechanical stability which allows the membraneimplant to be stretched over complex, three dimensional bone defectswithout tearing. Such materials are also suitable for suturing.

[0017] Advantageously, the barrier layer consists of long collagenfibres which are so closely connected that high molecular substancescannot permeate this barrier. The long fibres provide high tensilestrength and resistance to tearing so that the material is not only agood separation membrane but can also be readily sewn. It is importantin surgery that membrane implants can be sewn or pinned into positionand many of the membranes which have previously been proposed do notprovide this capability. The membrane in accordance with the inventionis mechanically stable enough to be handled surgically for implantation.

[0018] The matrix layer is very porous and may have a specific weight aslow as 0.02, which permits cells very rapidly to grow into this layer.This layer of the membrane, which normally also containsglycosaminoglycans, swells strongly and can take up as much as 5000% ofliquid. Ideally, the matrix layer should provide a pore structure (porevolume fraction and pore size) which allows cell adhesion and growth andwhich permits the seeded cells to maintain the chondrocytic phenotype,characterised by synthesis of cartilage-specific proteins. Pore sizeswill depend on the freeze drying process used to produce the collagen IImatrix but can be expected to be in the range of from 10 to 120 μm, e.g.20 to 100 μm. Optionally the pore size should be around 85 μm. Such apore size may readily be obtained by slow freezing at from −5 to −10° C.for about 24 hours followed by freeze-drying, or by adding ammoniumbicarbonate to the slurry before lyophilisation.

[0019] The matrix layer of the membrane is preferably provided bycollagen II material obtained from cartilage, preferably hyalinecartilage from pigs.

[0020] Whilst the desired thickness of the matrix layer will depend uponthe nature of the bone or chondral defect to be treated, in general thiscan be expected to be in the range of from 2 to 10 mm, e.g. from 4 to 6mm. The thickness of the barrier layer is preferably from 0.2 to 2 mm,e.g. from 0.2 to 0.7 mm.

[0021] The barrier layer may be provided by a natural animal membranecomprising collagen I and III. Being derived from a natural source, thisis totally resorbable in the body and does not form toxic degradationproducts. Such membranes also have particularly high tear strength ineither a wet or dry state and can therefore be surgically stitched ifnecessary. When moist the material is very elastic which allows this tobe stretched over irregularly shaped bone defects.

[0022] Besides collagen, natural animal membranes contain many otherbiomaterials, which must be removed. It is known to treat such membraneswith enzymes, solvents or other chemicals to effect purification and touse these membranes in medicine. Most of these materials are too thinand very often not particularly easy to use. The collagen fibrils havelost their native character and further disadvantages are that thematerial often has insufficient strength for use as a sewable material,has no water-swelling properties and provides no difference between thesmooth grain side and the fibrous flesh side. The fibrous form ofpurified telopeptide-free collagen Type I or II, being less soluble andbiodegradable, has been found to provide the most advantageous carriermaterial.

[0023] Membranes providing the barrier layer of the product according tothe invention include peritoneum membrane from calves or pigs whichretain their natural collagen structure. Peritoneum membranes from youngpigs aged 6-7 weeks old (weighing 60-80 kg) are especially preferred.

[0024] The barrier layer should preferably comprise pure, native (notdenatured) insoluble collagen and may be prepared in accordance with themethod described in WO-A-95/18638. The natural membrane may thus firstbe treated with alkali, for example aqueous NaOH at a concentration offrom 0.2-4% by weight. This serves to saponify any fats and alsoproteins which are sensitive to alkali. The second step is the treatmentof the material with an acid, usually an inorganic acid such as HCl.This eliminates acid-sensitive contaminants. The material issubsequently washed until the pH is in the range 2.5-3.5. The membranethen has a smooth or grain side and a looser more fibrous side. It maybe beneficial to effect some cross-linking of the membrane by heating to100-120° C.

[0025] The collagen II material used to provide the matrix layer of themembrane can be obtained from cartilage by a similar procedure to thatdescribed above in relation to the barrier layer comprisingpredominantly collagen I and III. It is preferable to remove water fromthe cartilage by treatment with acetone followed by extraction of fatwith a hydrocarbon solvent such as n-hexane, though alkanols such asethanol, ethers such as diethyl ether or chlorinated hydrocarbons suchas chloroform, or mixtures thereof may be used. The defatted material isthen subjected to treatment with alkali which saponifies any residualfat and degrades some of the proteins present. Finally, the material istreated with acid which effects further protein degradation. Thematerial is allowed to swell in water and is passed through a colloidmill to produce a slurry.

[0026] To produce the multi-layer membrane, the soft slurry containingcollagen II is applied to the fibrous side of the smooth membraneprepared, for example in accordance with WO-A-95/18638. Normally, themembrane will be placed on a smooth surface with the grain side down sothat the collagen II slurry can readily be applied, e.g. by rubbing intothe fibrous side of the membrane. The slurry thus forms a layer of anydesired thickness which firmly adheres to the collagen membrane. Thedouble-layer so formed is then subjected to freezing and freeze-dryingto provide the desired sponge-like structure having a desired pore size.If necessary, some of the matrix layer may be removed to provide adouble-membrane of uniform thickness. To produce a three-layer membrane,a second smooth membrane is then placed on top of the matrix layer withits fibrous side in contact with the matrix layer.

[0027] The collagen II slurry to be applied to the membrane in generalcontains 1.0-4.0 weight % of the collagen, advantageously 2-3 weight %.Conveniently, the pH value of this mixture should be adjusted to2.5-4.5, advantageously 3.0-4.0.

[0028] Advantageously, the collagen II material may be cross-linkedafter the freeze-drying step to stabilise the matrix layer. This alsoserves to increase the mechanical stability of the matrix layer and toreduce its rate of resorption by the body. Ideally, the degree ofcross-linking should be such that the rate of degradation of the matrixmatches the rate of tissue regeneration. Physically, cross-linking maybe carried out by heating, but this must be effected carefully to avoidundesired loss of resorbability. Heating to temperatures of 100-120° C.for a period of from 30 minutes to 5 hours is preferable. Morepreferably, cross-linking may be effected by UV irradiation using a UVlamp e.g. for a period of up to 8 hours.

[0029] The collagen II material advantageously containsglycosaminoglycans. The latter actually reacts with the collagen II toeffect some cross-linking and produces an insoluble product. Ifnecessary, further cross-linking can be effected by heating the materialor by UV irradiation as discussed above. The reaction between theglycosaminoglycans and the collagen can be effected at ambienttemperatures at a pH in the range 2.5-3.5. The quantity ofglycosaminoglycan may be between 1 and 10% by weight. The material maybe subjected to freezing and freeze-drying immediately after suchtreatment.

[0030] Alternatively, slurry formation may be effected by raising the pHof the collagen II mass. In this procedure, the mass is cooled to about4° C. and the pH value slowly raised by addition of cold aqueous NaOH at4° C. up to a pH value 6.5-7.5. Subsequently, the mass is held atambient temperature for 15-25 hours. In this time, the slurry is formedand after slurry formation, the mass can be frozen and freeze-dried.

[0031] A still further alternative is to neutralise the collagen II massto a pH value 6.8-7.4, subsequent to removal of air. The mixture isplaced in the mould and incubated for 15-20 hours at 37° C. A fineslurry develops which can subsequently be frozen and freeze-dried.

[0032] Which of the above three methods is used depends upon theproperties of the desired product. The first process gives the moststable product. However, the precipitation may give clumps of materialand must be very carefully carried out. The second method gives a softand uniform product which is, however, more soluble than the product ofthe first process.

[0033] In the production of the slurry, it is possible to additionallyintroduce further desirable substances such as medicines, e.g.antibacterials such as taurolidine or antibiotics such as gentamycin.

[0034] After the application of the slurry to the membrane, the materialis frozen. In order to obtain a reproducible pore size, the freezingmust be carefully controlled and the rate and time of freezing, the pHvalue and the particle size must be accurately controlled. In order toobtain very small pores, the material may be shock frozen at very lowtemperature.

[0035] The frozen membrane is then freeze-dried and subsequently heatedto 110-130° C. In this way, some cross-linking is effected.Subsequently, the freeze-dried biomembrane may be adjusted to therequired thickness so that the thickness of the matrix layer is commonlyabout 2 mm. The double membrane is then sterilised, for example bygamma-irradiation or with ethyleneoxide. Sterilisation by strongirradiation e.g. with ⁶⁰Co in doses of 25 kGy may deactivate the BMPs.In such circumstances, the sterile matrix may be impregnated with BMPsin sterile saline prior to implantation.

[0036] The membrane according to the invention can be used in medicinein the following ways:

[0037] As a material for guided tissue regeneration. Cell growth isencouraged by the matrix layer. The barrier layer inhibits undesiredcell growth.

[0038] As a material for the repair of chondral defects, i.e. lesionswhich do not penetrate the subchondral plate, and in the repair ofosteochondral defects.

[0039] The invention also provides the use of a multi-layer collagenmembrane as described above in guided tissue regeneration. The collagenII content of the membrane is particularly suitable for regeneration ofcartilage tissue but is also suitable for other tissue types.

[0040] Viewed from a further aspect the invention thus provides amembrane as hereinbefore described for use as a guided tissueregeneration implant.

[0041] The invention further provides a method of treating a bone orcartilage defect in the human or non-human animal body, said methodcomprising application of a membrane as hereinbefore described to thedefect, said membrane being oriented such that the barrier layerprevents the ingrowth of undesirable tissue types into the area of boneor cartilage regeneration.

[0042] The following examples are given by way of illustration only. Inthe Examples, all steps have to be performed under aseptic conditionsin, for example, Clean Rooms.

EXAMPLE 1

[0043] (A) Peritoneal membranes from young calves are completely freedfrom flesh and grease by mechanical means, washed under running waterand treated with 2% NaOH solution for twelve hours. The membranes arethen washed under running water and acidified with 0.5% HCl. After thematerial has been acidified through its entire thickness (about threehours) the material is washed until a pH of 3.5 is obtained. Thematerial is then shrunk with 7% saline solution, neutralised with 1%NaHCO₃ solution and washed under running water. The material is thendehydrated with acetone and degreased with n-hexane.

[0044] (B) Frozen cartilage from freshly slaughtered pigs was steeped incold water, thoroughly washed through and mechanically purified fromflesh residues, bones and hard pieces. Subsequently, the material waswashed for 30 minutes under flowing water.

[0045] Subsequently, the material was ground three times in ahomogenizer. The optical particle size at the end of size reduction wasabout 8 mm.

[0046] The cartilage pieces were dewatered by washing 4 times withacetone, each time for 8 hours. The cartilage was then defatted byextraction 4 times with n-hexane. Each treatment lasted at least 8hours. The ratio of hexane to cartilage was 1:10.

[0047] After defatting, the cartilage was swelled in drinking water. Theratio of water:material was 10:1. The treatment time was 24 hours.

[0048] The material was then treated with NaOH (5% by weight) wherebythe ratio of cartilage to liquid was 1:4 and the treatment time was 32hours. During the treatment, the pieces of cartilage were well stirred.Subsequently, the alkali was washed from the cartilage. The original pHof 14 was thereby reduced to 9-11. The dissolved impurities were washedout and separated from the cartilage. The liquid resulting from thealkaline treatment was collected for the recovery of glycosaminoglycan.

[0049] The collagen material was then treated with strong HCl (about 3%by weight) initially at a pH value under 1.0. The treatment time was 4-6hours.

[0050] Subsequently, the material was washed with cold water long enoughfor the pH value to rise to 3-3.5. All impurities were removed and theproduct was a salt-free collagen mass, suitable for production of asponge or other collagen material. For that purpose, the cartilage massmay be, according to the intended result, degassed, frozen andfreeze-dried.

[0051] The extract resulting from alkaline treatment above containedglycosaminoglycan, alkali, denatured proteins and salts. The extract wasfirstly neutralised with HCl, the pH value after neutralisation being 6.The extract was then treated with a filter aid, namely kieselguhr, whichhad the effect of removing the denatured proteins. 0.5 weight percent ofkieselguhr was introduced into the extract and removed by filtrationtogether with the denatured protein.

[0052] The supernatant was then submitted to ultrafiltration using amembrane having a molecular weight cut off at about 10,000 daltons. Inthis way, salts were removed to leave purified glycosaminoglycan.

[0053] The glycosaminoglycan solution so obtained was admixed withcollagen material from above to provide a collagen II matrix containingglycosaminoglycan.

[0054] The collagen II mass had the following properties:

[0055] TG=2.8 weight %

[0056] GAG=3 weight % (calculated on the basis of collagen)

[0057] pH value 3.5

[0058] (C) The freshly prepared peritoneum membrane prepared as in (A)above was uniformly soaked in water and laid flat on a glass plate withthe fibrous side upwards. Subsequently, the membrane was thoroughlywetted with the collagen II mass prepared as in (B) above. The membranewas stretched flat in all directions so as to remain adhered to theplate. The collagen II mass was thereby rubbed into the membrane.

[0059] The very thick mass was applied to the membrane and the plate wasleft overnight in the refrigerator at a temperature of about 4° C. Inthat period a slurry was formed.

[0060] The slurry was frozen under the following conditions: Temperatureof the bath −12° C. Time 40 minutes

[0061] Subsequently, the frozen slurry was freeze-dried and then warmedto 125° C. Freeze-drying time = 14 hours.

[0062] The collagen II matrix layer was subsequently split to athickness of 1 mm.

EXAMPLE 2

[0063] The freshly prepared peritoneum membrane from Example 1(A) wasapplied to a glass plate and the thick collagen II mass, having theproperties as in Example 1, was rubbed into the membrane. 50 g of thecollagen II mass was diluted to 100 ml with distilled water andthoroughly stirred. During the stirring, 100 ml of glycosaminoglycansolution was slowly added. Collagen was precipitated in the form of amass together with GAG. After the precipitation, the mass washomogenised and the dispersion so obtained was applied to the membrane.A slurry formed overnight and the treated membrane was further processedas described in Example 1.

1. A membrane comprising a resorbable multi-layer membrane for use invivo in the reconstruction of bone or cartilage tissue, said resorbablemulti-layer membrane comprising a matrix layer having a matrixconsisting essentially of collagen II and having an open sponge-liketexture, and at least one barrier layer having a close, relativelyimpermeable texture, the at least one barrier layer consistingessentially of a barrier layer material selected from the groupconsisting of collagen I, collagen III and a mixture thereof, whereinsaid multi-layer membrane is formed by adhering of said matrix layer tosaid at least one barrier layer by non-sutured adhesive, so that saidmatrix layer is adhered firmly to and in direct contact with said atleast one barrier layer.
 2. A membrane as claimed in claim 1 in whichthe matrix layer is provided between two barrier layers.
 3. A membraneas claimed in claim 1 in which the matrix layer is provided by collagenII material derived from natural cartilage.
 4. A membrane as claimed inclaim 3 wherein the collagen II material is derived from hyalinecartilage from pigs.
 5. A membrane as claimed in claim 3 in which thecollagen II material is physically cross-linked.
 6. A membrane asclaimed in claim 4 in which the at least one barrier layer is derivedfrom peritoneum membrane from calves or pigs.
 7. A membrane as claimedin claim 1 in which the matrix layer is impregnated with chondrocytesisolated from articular cartilage, periosteum, periocardium ormesenchymal stem cells from bone marrow.
 8. A membrane as claimed inclaim 1 in which the matrix layer, said at least one barrier layer, oreach said layer is impregnated with a glycosaminoglycan.
 9. A membraneas claimed in claim 8 wherein the glycosaminoglycan is hyaluronic acid,chondroitin 6-sulphate, keratin sulphate or dermatan sulphate.
 10. Amembrane as claimed in claim 1 in which the matrix and barrier layersare substantially free from proteoglycans.
 11. A membrane as claimed inclaim 1 in which the matrix layer, said at least one barrier layer, oreach of the layers further comprise chondronectin, lectin, fibronectin,calcium alginate, anchorin II, growth factors or bone morphogeneticfactors.
 12. A process for the preparation of a membrane as claimed inclaim 1 in which a collagen II slurry is applied to a surface of abarrier membrane having a close, relatively impermeable texture,followed by freeze-drying whereby to provide a matrix layer having anopen sponge-like structure.
 13. A method of treating a bone or cartilagedefect in the human or non-human animal body, said method comprisingapplication of a membrane as claimed in claim 1 to the defect, saidmembrane being oriented such that the barrier layer or layers preventthe ingrowth of undesirable tissue types into the area of bone orcartilage regeneration.
 14. A method of treating a bone or cartilagedefect in the human or non-human animal body, said method comprisingapplication of a membrane as claimed in claim 1 to the defect, saidmembrane being oriented such that the barrier layer or layers preventthe ingrowth of undesirable tissue types into the area of bone orcartilage regeneration, and wherein the matrix layer of said membrane isimpregnated with chondrocytes either immediately prior to or followingapplication to the defect.
 15. The method of claim 13 wherein saidmembrane is applied during arthroscopic surgery.
 16. The method of claim14 wherein said membrane is applied during arthroscopic surgery.
 17. Amembrane as claimed in claim 11 wherein the growth factors are cartilageinducing factor (CTF), insulin-like growth factor (IGF), or transforminggrowth factor (TGF ).
 18. A membrane as claimed in claim 11 wherein thebone morphogenetic factors are human BMP-2, BMP-3, BMP-4 or BMP-7.
 19. Amembrane comprising a resorbable multi-layer membrane for use in vivo inthe reconstruction of bone or cartilage tissue at a site of a defect insaid tissue, said resorbable multi-layer membrane comprising a matrixlayer having a matrix comprising collagen I, collagen III or a mixturethereof, and having an open sponge-like texture, and at least onebarrier layer having a close, relatively impermeable texture, the atleast one barrier layer comprising a barrier layer comprising collagenI, collagen III or a mixture thereof, wherein said multi-layer membraneis formed by application of said matrix layer to said at least onebarrier layer as a slurry, so that said matrix layer is adhered firmlyto and in direct contact with said at least one barrier layer, whereinfirm adherence of the matrix layer to said at least one barrier layerresults essentially solely from application of said slurry to said atleast one barrier layer.
 20. A membrane comprising a resorbablemulti-layer membrane for use in vivo in the reconstruction of bone orcartilage tissue at a site of a defect in said tissue, said resorbablemulti-layer membrane comprising a matrix layer having a matrixcomprising collagen I, collagen II or a mixture thereof, and having anopen sponge-like texture, and at least one barrier layer having a close,relatively impermeable texture, the at least one barrier layercomprising collagen I, collagen III or a mixture thereof, wherein saidmulti-layer membrane is formed by application of said matrix layer tosaid at least one barrier layer by non-sutured adhesive, so that saidmatrix layer is adhered firmly to and in direct contact with said atleast one barrier layer.